On-line optical and X-ray spectroscopies with crystallography: an integrated approach for determining metalloprotein structures in functionally well defined states

Abstract

X-ray-induced redox changes can lead to incorrect assignments of the functional states of metals in metalloprotein crystals. The need for on-line monitoring of the status of metal ions (and other chromophores) during protein crystallography experiments is of growing importance with the use of intense synchrotron X-ray beams. Significant efforts are therefore being made worldwide to combine different spectroscopies in parallel with X-ray crystallographic data collection. Here the implementation and utilization of optical and X-ray absorption spectroscopies on the modern macromolecular crystallography (MX) beamline 10, at the SRS, Daresbury Laboratory, is described. This beamline is equipped with a dedicated monolithic energy-dispersive X-ray fluorescence detector, allowing X-ray absorption spectroscopy (XAS) measurements to be made in situ on the same crystal used to record the diffraction data. In addition, an optical microspectrophotometer has been incorporated on the beamline, thus facilitating combined MX, XAS and optical spectroscopic measurements. By uniting these techniques it is also possible to monitor the status of optically active and optically silent metal centres present in a crystal at the same time. This unique capability has been applied to observe the results of crystallographic data collection on crystals of nitrite reductase from Alcaligenes xylosoxidans, which contains both type-1 and type-2 Cu centres. It is found that the type-1 Cu centre photoreduces quickly, resulting in the loss of the 595 nm peak in the optical spectrum, while the type-2 Cu centre remains in the oxidized state over a much longer time period, for which independent confirmation is provided by XAS data as this centre has an optical spectrum which is barely detectable using microspectrophotometry. This example clearly demonstrates the importance of using two on-line methods, spectroscopy and XAS, for identifying well defined redox states of metalloproteins during crystallographic data collection.

Towards accurate structural characterization of metal centres in protein crystals: the structures of Ni and Cu T6bovine insulin derivatives

“…The development of multifunctional beamlines which combine macromolecular crystallography with spectroscopy has facilitated single-crystal spectroscopy experiments on protein crystals with concurrent collection of crystallographic data (Antonyuk & Hough, 2011;Pearson & Owen, 2009;De la Mora-Rey & Wilmot, 2007). Among the spectroscopies used in combination with XRD are X-ray absorption spectroscopy (XAS; Arcovito & della Longa, 2012; Cotelesage et al, 2012; Yano & Yachandra, 2008;Strange et al, 2005;Hasnain & Strange, 2003), UV-visible absorption spectroscopy (Hersleth & Andersson, 2011;Ellis et al, 2008;Pearson et al, 2004) and Raman spectroscopy (Katona et al, 2007;McGeehan et al, 2011;Hersleth & Andersson, 2011).…”

Current advances in synchrotron radiation instrumentation for MX experiments

“…Several beamlines offer the acquisition of optical spectroscopic data concurrent with the collection of diffraction data sets (Carpentier et al , 2007, Davies et al , 2009, Ellis et al , 2008, Owen et al , 2009, Pearson et al , 2007, Pompidor et al , 2013, Royant et al , 2007, Sakai et al , 2002, Stoner-Ma et al , 2010, Cohen et al , 2016). Key applications of this complementary information are the identification of kinetic reaction intermediates in structural enzymology, as well as the quantitative monitoring of changing oxidation states of metal centers due to X-ray induced photoelectrons.…”

Cryoradiolysis as a Method for Mechanistic Studies in Inorganic Biochemistry

“…170 Sato et al observed almost complete reduction of the high molecular weight cytochrome c from Desulfovibrio vulgaris during data collection. 181,188 Spectroscopic results revealed that during X-ray data collection, one Cu center is reduced, while the other is not, and no internal electron transfer happens between these two Cu atoms. 181,188 Spectroscopic results revealed that during X-ray data collection, one Cu center is reduced, while the other is not, and no internal electron transfer happens between these two Cu atoms.…”